Pumpkin seeds (Cucurbita pepo subsp. ovifera) decoction promotes Trichinella spiralis expulsion during intestinal phase via “Weep and Sweep” mechanism

Trichinosis is a zoonotic disease of communal health concern as it instigated human outbreaks in several countries. Besides, the development of resistance, traditional therapy has numerous antagonistic effects. Thereby, finding efficient natural alternatives is required. In comparison to albendazole, this study evaluated the impact of pumpkin decoction on Trichinella spiralis in experimentally infected mice. The anthelmintic action of pumpkin decoction (500 mg/kg) was determined using T. spiralis infected mice in enteric phase for 5 days. Pumpkin decoction anthelmintic activity fortified by mixing with honey (1:1). Pumpkin decoction and Pumpkin decoction-honey mixture were evaluated by comprising with reference drug, albendazole (50 mg/kg). The T. spiralis adult count was significantly lower in all treated groups, with the pumpkin decoction-honey mixture showing the largest reduction (83.2%) when compared to the infected group (P ≤ 0.001). The intestinal histological changes and the level of COX-2 expression in the intestinal tissue were both significantly reduced in the same group. The pumpkin decoction improved the immune response, as evidenced by a significant decrease in nitric oxide (NO) and tumor necrosis factor (TNF-α) and a significant increase in the expression of the transforming growth factor (TGF-1β) and interleukin-17 (IL-17). The pumpkin decoction's anthelmintic action was facilitated by the TGF-1β and IL-17-driven Weep and Sweep mechanism. Both administration of pumpkin decoction beside honey showed the best treatment group that resulted in high infection reduction besides amelioration of biochemical markers and restoration of histological to normal state. In conclusion, pumpkin decoction is highly effective against T. spiralis which could be a promising alternative herbal drug and the pumpkin decoction effect was higher in the case of combination with honey.

Almost 4 million people are currently infected with helminthic parasites worldwide, which are a serious challenge for both health and the economy 1 .The distribution of nematode parasites from the genus Trichinella is widespread.Trichinellosis is a zoonotic parasitic illness that has been attributed to 12 different species and can infect a range of hosts, including humans 2 .In the intestine, when Trichinella spiralis larvae (L1) are digested from meat in the stomach, they travel to the intestinal tract and infiltrate epithelial cells.From L1 to adulthood, parasites develop in the intestine.For several weeks, adults reside inside intestinal epithelial cells, where they reproduce, and the female worms discharge newborn larvae (NBL) into the affected tissue.These NBL spread throughout the body, penetrating skeletal muscle cells; after that, they evolve into L1 that spread infection to the subsequent host 3 .According to the previous findings 4 , adults of T. spiralis are then evacuated from the intestine within 10-17 dpi; thus, the first 17 dpi might be regarded as the intestinal phase 5 .It has been demonstrated that having worms in the bowel causes a number of pathological alterations that cause acute inflammatory reactions

Authentication the plant and extraction
Cucurbita pepo L. subsp.ovifera (pumpkin) seeds were obtained from the Cucurbit and Cross-pollinated Vegetable Department, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt.The plant material was collected in compliance with relevant institutional, national, and international guidelines and legislation.The plant was identified by Prof. Dr. Abd El-Halim Abd El-Mogali Mohamed, Chief Researches, Flora & Phytotaxonomy Research Department, HRI, ARC.A voucher specimen (M-215) has been deposited in the herbarium of National Research Centre (CAIRC).The plant was kindly authenticated by Prof Dr. Mona Marzouk, Professor of Taxonomy, National Research Center, Cairo, Egypt.
Pumpkin seeds' decoction was performed by boiling 250 g from seeds without husk of pumpkin with distilled water at 100 °C three times each for one h (3 × 1L).Water was removed from filtrated solution by a Rotary evaporator to yield 98.25 g of pumpkin decoction.
Honey in the current study was Citrus honey, which was obtained from El-Sharkia Governorate, Egypt.The mixture of pumpkin decoction and honey was prepared by mixing equal volumes of two materials.

The impact of pumpkin decoction and its mixture with honey against T. spiralis worms
Selection and animal maintenance Sixty male Swiss albino strain (adult with 8-10 weeks old), weighing about 25-30 g were obtained from Theodor Bilharz Research Institute (TBRI) (Giza, Egypt).
The animal house was well ventilated and maintained at room temperature from 20-25 °C, 50-55% relative humidity, and a 12 h dark/light cycle.Mice were housed in large spacious hygienic cages during the experimental period, and they were provided with a pellet diet and water ad libitum.Pellet diet was obtained from the animal house of TBRI.

Induction
The mice were infected orally with about 300 larvae per mouse.The strain of T. spiralis was obtained from (TBRI).Each mouse received roughly 300 larvae by oral infection.The diaphragms of infected mice were where the strain of T. spiralis was first isolated.Mice with a high level of T. spiralis infection had their diaphragms cut and digested in a warm tap water solution made of 1% pepsin and 1% strong hydrochloric acid 27 .Larvae were extracted using the sedimentation method after an overnight incubation at 37 °C.They were then rinsed with saline multiple times, and the quantity of larvae/ml was measured using a hemocytometer.Using a tuberculin syringe, 300 larvae were injected orally into the 12-h-fasting mice 28 .
The dosing protocol of pumpkin decoction, honey, and standard drug Pumpkin decoction was investigated at a dose of 500 mg/kg/day for 5 days (0.10 of LD 50 ) according to Garg et al. 29 and Ghosh 30 .Also, honey was tested at 500 mg/kg/day for 5 days.The tested dose of pumpkin decoction and honey mixture was 500 mg/kg/day for 5 days.The reference drug, Albendazole was tested at its recommended dose; 50 mg/kg/day 31 and received from the 2nd dpi for 5 successive days, orally via gastric tube.

Experimental layout
After adaptation period (seven days), 60 adult male albino mice were classified into 3 main groups as follows: The first main group: 10 mice without infection and were orally administered saline solution along experimental period, these mice were kept as uninfected group.
The second main group: 10 mice were infected by T. spiralis larvae and were orally administrated saline solution along experimental period, these mice kept as infected group.
The third main group: 40 mice were infected by T. spiralis larvae and then were divided into four subgroups as follows: subgroup I: 10 infected mice were orally administrated pumpkin decoction at 500 mg/kg/ day.Subgroup II; 10 mice were administered honey at 500 mg/kg/ day.Subgroup III: 10 infected mice were orally administered pumpkin decoction-honey mixture at 500 mg/kg/day.Subgroup IV: 10 infected mice were orally administered Albendazole, reference drug at recommended dose 50 mg/kg/day; these mice were kept as standard group.

Collection of blood samples and intestine
At the end of the experiment, the animals were fasted over-night and intraperitoneally injected with 30 µL/mouse (weighted about 15 g) 32 of the combination of 87 mg ketamine/ kg and 13 mg/ xylazine of body weight dissolved in normal saline.Achieving anesthesia started 10-15 min after simultaneous injection and continued to 15-30 min.The blood samples were collected from the retro-orbital plexus, and serum was obtained by centrifugation at 4000 rpm for 10 min using Sigma Laborzentrifugen (Osterode am Harz, Germany).Anesthetic animals were sacrificed by cervical dislocation after that the intestine was collected, washed, and weighed.Intestine 10% homogenate in phosphate buffer (pH,7) was prepared using an ultrasonic tissue homogenizer.

Assessment of adult worm count in small intestine
The small intestines of the sacrificed mice were removed, opened longitudinally, washed with saline, and sectioned into small pieces and incubated at 37°C in phosphate buffered saline (PBS) for 2 h then washed 3 times, taking into account that all the solutions from the previous step were collected to be centrifuged for 3 min at 2000 rpm 33 .
The number of adults in the sediment was counted under an inverted microscope using low power magnification × 100 (Leica DM3000, Houston, Texas).

Biochemical analysis
Antioxidant and oxidative stress biomarkers were determined spectrophotometrically (JASCO V-630 Spectrophotometer serial No. C317961148) in the intestine homogenate.Glutathione (GSH) concentration was determined according to the method of Griffith 34 .Antioxidants enzyme activities, including glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), were determined according to the methods of Goldberg and Spooner 35 , Habig et al. 36 , Fridovich 37 and Beers and Sizer 38 , respectively.
Oxidative stress biomarkers, including nitric oxide (NO) and malondialdehyde (MDA), were measured according to Montgomery and Dymock 39 and Ohkawa et al. 40 .

Histopathological examination
To detect histopathological changes of Intestinal tissue, ileal specimens were dissected out, opened longitudinally, washed and one piece of the last third of all mice were stored in 10% neutral buffered formalin.Then, they dehydrated, cleared and embedded in paraffin blocks.Paraffin sections were cut to be of 5 μm thickness, stained with haematoxylin and eosin (H& E) 45 .To quantify the number of the goblet cells, four non-overlapping fields were visualized and acquired using Olympus BX63 microscope image system (Olympus, Tokyo, Japan) under high power field (magnification of 400×) analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD, USA).

Immunohistochemical detection and evaluation of COX-2
Immunohistochemical staining was carried out using the primary anti-Cyclooxygenase-2 antibody (COX-2) (ab15191, 1/100, rabbit polyclonal antibody, Abcam, USA), species specificity including mice).Heat-mediated antigen retrieval and standard labeled streptavidin-biotin immunoenzymatic antigen detection procedure was performed according to Aboulhoda and Abd El Fattah 46 .Mayer's hematoxylin was then used to counterstain the sections.Four non-repeating immunohistochemical images of each sample were visualized and acquired using Olympus BX63 microscope image system (Olympus, Tokyo, Japan) under high power field (magnification of 400×).Quantitative estimation of immunohistochemical staining based on the determination of the % positivestained area analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD, USA).

Determination of the chemical composition of pumpkin decoction
Quantitative analysis of pumpkin decoction Total lipid content was determined according to A.O.A.C. 47 and was expressed as mg lipid/ g pumpkin decoction.Total phenol content was estimated by Folin-Ciocalteau's reagent according to Singleton et al. 48and was described as mg gallic acid /g pumpkin decoction.Flavonoid concetration (mg quercetin /g) was determined as Lin and Tang 49 .Total alkaloids content was gravimetrically determined according to Onwuka 50 and was expressed as mg alkaloids /g pumpkin decoction.Tannins content was measured by Folin-Ciocalteau's reagent according to Ranganna 51 and was expressed as mg tannic acid/ g pumpkin decoction.Total saponins content was gravimetrically determined according to Edeoga et al. 52 and was expressed as mg saponins/ g pumpkin decoction.
Identification of the phenolic composition of pumpkin decoction using HPLC analysis An Agilent 1260 series was used for the HPLC analysis.Eclipse C18 column (4.6 mm × 250 mm i.d., 5 μm), was used for the separation.The mobile phase consisted of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate of 0.9 ml/min.The linear gradient was sequentially programmed into the mobile phase as follows: 0 min (82% A), 0-5 min (80% A), 5-8 min (60% A), 8-12 min (60% A), 12-15 min (82% A); 15-16 min (82% A) and 16-20 (82%A).The multi-wavelength detector was seen at 280 nm.For each of the sample solutions, 5 μl of injection volume was used.The column was kept at a constant temperature of 40 °C.

Identification of lipid composition of pumpkin decoction using GC analysis (A) Fatty acids composition
The GC model 7890B from Agilent Technologies was equipped with flame ionization detector at Central Laboratories Network, National Research Centre, and Cairo, Egypt.A Zebron ZB-FAME column (60 m × 0.25 mm internal diameter × 0.25 μm film thickness) was used to obtain the separation.Hydrogen was served as the carrier gas in the analysis with a flow rate of 1.8 ml/min in split-1:50 mode, an injection volume of 1µ l, and the following temperature program: 100 °C for 3 min; rising at 2.5 °C/ min to 240 °C and held for 10 min.The injector and detector (flame ionization detector, FID) were held at 250 °C and 285 °C, respectively.
(B) Unsaponifiable matter composition Sample preparation: The lipids of sample were saponified with ethanolic potassium hydroxide, unsaponifiable fraction extracted with ether and petroleum ether 1:1(v/v) and evaporated at 40°C.

Sample derivatization:
The unsaponifiable fraction extracted with ether and petroleum ether (1:1) was mixed with 50 µL of bis(trimethylsilyl)trifluoroacetamide (BSTFA) + trimethylchloro-silane (TMCS) 99:1 silylation reagent and 50 µL pyridine for derivatization sample functional groups to trimethylsilyl groups (abbreviated TMS) prior to GC analysis.The Central Laboratories Network, National Research Centre, Cairo, Egypt's mass spectrometer detector (5977A) and gas chromatograph (7890B) were both part of the GC-MS system (Agilent Technologies).The GC was outfitted with an HP-5MS column that measured 30 m × 0.25 mm internal diameter and film thickness of µm.Analysis was performed by hydrogen as the carrier gas at a flow rate of 2.0 ml/min at a split 1:20, injection volume of 1 µl and the following temperature program: 50 °C for 5 min; rising at 5 °C /min to 100 °C and held for 0 min and rising at 10 °C /min to 320 °C and held for 10 min.The injector and detector were held at 280 °C, 320 °C.Mass spectra were obtained by electron ionization (EI) at 70 eV; using a spectral range of m/z 25-700 and solvent delay 6 min.The mass temperature was 230°C and Quad 150 °C.By comparing the spectrum fragmentation pattern with those found in the Wiley and NIST Mass Spectral Library data, it was possible to identify various ingredients.

Statistical analysis
Data were analyzed as mean ± SE for six mice each.Comparisons among groups were performed by one-way analysis of variance ANOVA test at P ≤ 0.001 and P ≤ 0.05 followed by Tukey comparison test using IBM-SPSS (version 25) followed by a post hoc test.

Anti-trichinosis effect of pumpkin decoction and its mixture with honey in trichinosis
Table 1 summarises the effects of plant decoction on the percentage reduction of the adult stage of T. spiralis in mice.Treatment with pumpkin decoction showed a significant reduction in the recovery rate of adult worms by 69.6%.However, a significant decrease of 37.03% in the recovery rate of adult worms was observed in the case of treatment with honey.Treatment with pumpkin and honey resulted in a high elimination rate of worms (83.2%), whereas treatment with a 50 mg/kg dose of ALB showed a 50.3% decrease in the worm recovery rate.

Antioxidant effect of pumpkin decoction and its mixture with honey in trichinosis.
Data in Table 2 present the effect of pumpkin decoction, honey, and their mixture on antioxidant system of trichinosis mice, compared to infected mice (without treatment).The effect of the pumpkin decoction on trichinosis was evaluated by comparison with standard drug, albendazole.
Table 1.Anthelmintic efficacy of pumpkin seed decoction and its mixture with honey against adult stage of T. spiralis infection in mice.www.nature.com/scientificreports/ Trichinosis caused considered oxidative stress that represented as a significant depletion in GSH concentration (19.58%) concurrent with significant inhibition in the activity of GR (31.95%),GST (32.56%),CAT (26.46%), and SOD (39.67%) in comparison to the uninfected group (P ≤ 0.001).
On the contrary, GSH level and its related enzymes; GR and GST significantly elevated when trichinosis mice were force fed pumpkin decoction (500 mg/kg for 5 days), which increased by about 61.62, 76.53, and 101.90, respectively, compared to the infected group (P ≤ 0.001).Also, there are no significant differences recorded between the effect of pumpkin decoction and honey on GSH and related enzymes.Interestingly, GSH and GR of the pumpkin decoction group or honey group were equal to the reference drug group, meanwhile GST was higher than that of reference drug group.
Both of CAT and SOD activities of trichinosis mice were activated significantly by administration pumpkin decoction and elevated by about 70.14 and 25.62%, respectively in comparison to the infected group (P ≤ 0.001).In addition, the effect of pumpkin decoction on CAT and SOD was higher than the effect of honey and the reference drug.
Data in Table 3 present the effect of pumpkin decoction, honey, and their mix on the intestinal NO concentrations of trichinosis mice, compared to infected mice without treatment.Trichinosis induced NO production significantly, which was higher than the uninfected group by about 130.97% (P ≤ 0.001).Compared to infected group, pumpkin decoction or honey and their mix did not record significant effect on NO production.On the contrary, standaed drug significantly reduced NO production in comparison to infected control (P ≤ 0.001).
MDA, an oxidative biomarker, is the end product of lipid peroxidation.Data in Table 3 indicated that lipid peroxidation was promoted as a response to trichinosis, which represented a significant increase in MDA levels of infected group (5.47 ± 0.22 nmol/ g) in comparison to uninfected group (2.75 ± 0.18 nmol/ g).Meanwhile, all treated groups recorded a considerable reduction in MDA levels.Pumpkin decoction-honey mixture caused the highest reduction on MDA; 2.76 ± 0.19 nmol/ g versus 5.47 ± 0.22 nmol/ g in the infected group (P ≤ 0.001).Mixture of pumpkin decoction and honey returned MDA to the value of the uninfected group.

Immune modulatory effect of pumpkin decoction and its mixture with honey in trichinosis.
Figure 1 shows that trichinosis promoted macrophages and their phagocytosis as evident by the significant elevation on its secretory product, TNF-α.In the infected group, TNF-α expression was elevated to 0.890 ± 0.01 ng/g compared to the uninfected group: 0.760 ± 0.01 ng/g (P ≤ 0.001).Pumpkin decoction or honey significantly suppressed TNF-α expression in these groups (0.674 ± 0.02 and 0.846 ± 0.009 ng/g, respectively), in comparison to the infected group (0.890 ± 0.01 ng/g), P ≤ 0.001.Mixing pumpkin decoction with honey did not improve its activity significantly.Meanwhile, the lowest TNF-α expression was recorded in reference drug group.The effect of all tested materials on TNF-α is predominantly suppressive.
Figure 2 shows that T. spiralis infection induced TGF-β1 expression in the infected control (6.56 ± 0.29 ng/ml) more than the uninfected control (4.51 ± 0.38 ng/ml), (P ≤ 0.001).More of induction for TGF-β1 was happened when infected mice administrated pumpkin decoction (10.13 ± 0.34 ng/ml), honey (8.41 ± 0.38 ng/ml), compared to the infected control.The effect of pumpkin + honey mixture did not differ significantly than pumpkin decoction.All studied materials showed stimulating effect on TGF-β1.
Figure 3 represents that T. spiralis infection promoted T helper 17 cells differentiation that evident as s significant augment in IL-17 expression in the infected control (651.64 ± 8.08 pg/g), which was higher than the uninfected control by about two folds (P ≤ 0.001).Treating by pumpkin decoction or honey significantly increased the induction of IL-17, and the effect of pumpkin decoction (733.04 ± 6.26 pg/g) was higher than honey (686.76 ± 5.37 pg/g), compared to the infected group (P ≤ 0.001).Mixing pumpkin decoction and honey increased IL-17 expression more than pumpkin deccotion or honey and recorded the highest IL-17 level (757.86 ± 6.26 pg/g).On the contrary, reference drug significantly inhibited IL-17 expression (503.21 ± 2.68 pg/g) to be lower than the infected control (651.64 ± 8.08 pg/g).All studied materials showed stimulating effect on IL-17, except the reference drug.

The effect of pumpkin decoction and its mixture with honey on safety profile of trichinosis:
To recommend the pumpkin decoction and its mixture with honey as a repellent drug for T. spiralis, the safety profile of the pumpkin decoction and its mixture must be evaluated during experimental period.Therefore, in this study, we assessed the toxicity of the pumpkin decoction and its mixture through its effect liver functions and compared these with that of the uninfected groups.
T. spiralis infection caused marked hepatic disruption, where liver produced low amount of protein, albumin, and globulin.As well as hepatocytes released high amount from liver enzymes, AST and ALT to blood stream as seen in Table 4. Compared to the uninfected control, T. spiralis infection significantly reduced TP (29.70%), albumin (34.64%), and globulin (17.50%), meanwhile it significantly elevated AST (280.90%) and ALT (33.64%).Inversely, all tested materials returned liver performance of infected mice that administered pumpkin, honey, and their mixture or reference drug towards normalization.The pumpkin group recorded the best protein profile, which was bitter than the uninfected group.Pumpkin significantly increased globulin concentration followed by honey and their mixture by about 155, 105.83, and 95.83%, respectively than the uninfected group (P ≤ 0.001).Also, all tested materials prevented infiltration of liver enzymes, AST and ALT into blood stream.

The effect of pumpkin decoction and its mixture with honey on histopathological examinations.
Examination of H&E-stained intestinal tissue from the healthy group revealed the same typical histological construction which consists of the mucosa, submucosa, muscularis externa, and serosa.The mucosa has three layers, including the epithelium, lamina propria, and muscularis mucosae, and is organised into villi.They are lined with columnar epithelium and numerous goblet cells (Fig. 4a,b).The infected non-treated group displayed an extensive inflammation was mostly in the center of the villi and extended into the submucosa.The villi were flattened and sloughed.T. spiralis adults could also be identified by the cut sections (Fig. 4c,d).In comparison to the infected, untreated group, the pumpkin decoction-treated group had a modest inflammation and an enhancement in the intestinal structure and villi' length (Fig. 4e,f).Otherwise, those who received honey or ALB treatment showed a considerable decrease in the inflammation severity and protracted villi (Fig. 4g,h,k,l).Mice treated with pumpkin decoction and honey showed intact intestinal villi with normal brush border.Furthermore, goblet cells showed hyperplasia (Fig. 4i,j).

The effect of pumpkin decoction and its mixture with honey on immunohistochemical results
The analysis of small intestinal tissue samples from an ideal control showed negative expression of COX-2 (Fig. 5a).While marked expression of COX-2 was observed in the positive control group (Fig. 5b).The administration of pumpkin decoction, or honey alone, or albendazole displayed moderate expression of COX-2 in    www.nature.com/scientificreports/ileal sections (Fig. 5c,d,f).However, pumpkin decoction with honey mixture substantially diminished COX-2 expression in the portions of small intestinal (Fig. 5e).

Chemical composition of pumpkin decoction:
To discus the mode of action of the pumpkin decoction, we determined the chemical composition of the pumpkin decoction.Pumpkin decoction (30.00 g/ 100 g pumpkin seeds powder) is an oil-like condition with oil-like odor and light-green color (Table 5).The quantitative determination of pumpkin decoction showed that it contains crude lipid (396.60 ± 12.25 mg lipid/ g pumpkin decoction), total phenolics compounds (80.65 ± 1.85 mg gallic acid /g pumpkin decoction), total flavonoids (52.00 ± 4.30 mg quercetin /g pumpkin decoction), total tannins (18.08 ± 0.45 mg tannic acid /g pumpkin decoction) crude saponins presented as 33.05 ± 1.63 mg saponin/ g pumpkin decoction and finally total alkaloids represented 41.47 ± 1.65 mg alkaloid/ g pumpkin decoction.

Discussion
Albendazole is commonly used versus intestinal parasites as its broad-spectrum activity and low cost 53 .Nevertheless, its efficacy is prompted by numerous factors, for instance oral bioavailability, that predominantly rely on the solubility, therapy dosage and the host biotransformation as well as time of onset of treatment after infection.That has inspired researchers to seek out novel, safe and potent anthelminthic agents 54 .The utilization of medicinal plants and the repurposing of currently accessible pharmaceuticals have emerged as two promising strategies in search for new therapeutic options 55,56 .
In the current study, we demonstrated that the therapeutic potential of pumpkin seeds decoction in-vivo during Trichinellosis intestinal phase in comparison to albendazole, as a reference drug.Our results demonstrated that pumpkin decoction significantly minimized the number of parasites in intestine by about 69.60%, compared to the infected group.Many plant extracts can reduce intestinal adult burden of T. spiralis including, Balanites aegyptica fruits 57 , myrrh (Commiphora molmol) and, thyme (Thymus vulgaris L.) 58 , and Punica granatum peel 31 .Also, many plant extracts showed toxic effect on T. spiralis in vitro as Lipidium sativum (garden cress), and Commiphora molmol (myrrh) 59 .In addition, Cucurbita maxima seeds (decoction or ethanolic extracts) have high anthelmintic activity against different parasites such as Aspiculuris tetraptera in naturally infected mice 60 .Also, Rupa and Jayanta 61 reported that CuCurbita maxima seeds have anthelmintic effect against earthworms (Pheretina posthumal).Similarly, another species of Cucurbita, Cucurbita pepo L, has strong anthelmintic activity against Ascaris suum 16 .But this study is the first study on the effect of Cucurbita pepo on Trichinella spiralis intestinal phase.
To increase the anthelminthic effect of the pumpkin decoction, we fortified it with honey (1:1).The antihelminthic effect of pumpkin + honey mixture was better than all of pumpkin, honey and albendazole.Mixture significantly reduced the number of parasites by about 83.20% relative to the infected group.This finding is consistent with the previous studies also elucidated about immunomodulatory properties of honey which significantly affect the production of cytokine, i.e., tumor necrosis factor-α thus, plays an important role in innate immunity in rats infected with T. gondii, Giardia and Trichomonas under in vitro conditions which is consistent with other natural products which enhances the immune system by participating as immune modulatory agents, respectively 62,63 .
Pumpkin seeds decoction and its mixture with honey caused significant immune responses as a significant elevation on TGF-1β and IL-17, and a significant reduction on TNF-α.Additionally, T. spiralis infection associated with oxidative stress status as a significant reduction on intestinal GSH concentrations and activities of antioxidant enzymes, GR, GST, CAT, and SOD, concurrent with a significant augment in intestinal NO and MDA concentration relative to the status in the uninfected group.Pumpkin decoction ameliorated oxidation system of Trichinellosis mice and significantly reduced oxidative stress biomarkers; NO and MDA concentrations and significantly induced antioxidant biomarkers: GSH, GR, GST, CAT, and SOD.The antioxidant capacities of pumpkin decoction were significantly improved when mixed with honey.
To explore the mode of action of anthelminthic activity of pumpkin seeds decoction and its mixture with honey, we studied their effect on immunity; type 1 (TNF-α and NO) and type 2 (TGF-1β and IL-17).Additionally, their role on the Weep and Sweep mechanism, the main mechanism in larvae expulsion.So, we determined two cytokines: TGF-β1, and IL-17 in intestinal tissues.
Firstly, in contrast to every disease model discussed thus far, it is type 2 immunity rather than type 1 immunity that protects mammals from helminths that was demonstrated by the following: i) Balb/C mice that tend to the type 2 immune responses are inherently susceptible to bacterial infections and are inherently resistant to worm infection, ii) inversely, mice prone to type 1 responses are innately resistant to most bacteria, are inherently susceptible to helminthic infections.Activated Mast cell has an important role in type 2 immunity against several helminths like Trichinella and Nippostrongylus.IL-9 is a key step for activation of mast cell that elevates gastrointestinal peristalsis, which successfully expels parasites outside the gut, which was confirmed as transgenic mice overexpressing IL-9 were extremely resistant to Trichinella infection and blocking of IL-9 exacerbated infection in mice normally resistant to Trichinella infection) 64,65 .Our results were in accordance with these facts, where pumpkin decoction and its mixture with honey significantly suppressed TNF-α and NO concurrently stimulated TGF-1β and IL-17.TNF-α as a biomarker of Type 1 immune response, primarily produced by macrophages was decreased significantly by pumpkin and pumpkin-honey mix, respectively.NO, the product of phagocytes (monocytes, macrophages, and neutrophils), type 1 immunity was remarked decreased in treated groups with pumpkin and pumpkin-honey mix, respectively.On the contrary, TGF-1β was considered as an anti-inflammatory cytokine previously, but presently, it was reported as a key step in Th17 differentiation.TGF-1β plays an important role in controlling the immune system.Most leucocytes cells secrete TGF-1β 66,67 .Covalently, we can conclude that pumpkin decoction and its mixture with honey inhibit Type 1 immunity and induce Type 2 immunity.
Secondly, to remove gastro-intestinal helminths an integrated response including the nervous, endocrine, and immune systems are used to expel the parasites.This is achieved by rising intestinal hydration and muscle contraction, which causes the worms to separate from the intestine and be released outside the body as part of a "Weep and Sweep" reaction 8 .
To explore the Weep stage, we explained the role of gastrointestinal tract (GI) in immune response.The GI area is a very particular site for immune system activity.Mucus is a physical, gel-like barrier that protects epithelium from microorganisms and gut contents.The physical capture of worms, which allows for their evacuation after detaching from tissues and entering the gut lumen, is the most basic mucus-dependent mechanism for aiding infection termination.Mucus is involved in weep stage; this mucus prevents the worms from attaching themselves to the intestinal wall to facilitate their expulsion 68,69  www.nature.com/scientificreports/TGF-1β, with the cytokine controlling mast cell expression of the gut homing integrin alpha E and MMCP-1, essential for the Weep aspects of T. spiralis 70 .Goblet cell hyperplasia is a crucial sign of intestinal nematode infection during the intestinal phase 71 .In mice infected with T. spiralis, the number of goblet cells rose to 7, 10, and 15 dpi 72 .Analogous outcomes have been attained in mice infected with T. spiralis at 10 dpi 73 .In comparison to the control group, the goblet cell numbers in T. spiralis-infected mice spontaneously rose at 7 and 10 dpi, and this number was higher at 10 dpi than at 7 dpi 74 .These outcomes are all comparable to what our investigation revealed.Mucus is one of the active molecules secreted by goblet cells, which is known to physically shield the mucosal surfaces of the small intestine.Several investigations have revealed that T. spiralis-infected mice's small intestines overexpress both the membranebound Muc-3 and the secreted Muc-2.It has been demonstrated through the use of multiple experimental models that Th2 cytokines are necessary for goblet cell hyperplasia 4,75 .
Goblet cell hyperplasia is also largely dependent on stimulation of the IL-4/IL-13 receptor, IL-4Ra, and the activation of downstream STAT6.In Stat6 or IL-4Ra deficient mice infected with T. spiralis, goblet cell hyperplasia has not been detected, hindering worm expulsion 76 .Thereby has been proposed that the goblet cell hyperplasia seen in T. spiralis infections is a mucosal epithelial response to Th2 cytokines, primarily regulated by IL-13 77 .Activated CD4 + cells can differentiate into Th1, Th2, Th17, or Treg subset populations in response to antigenic stimulation.The cytokines in the microenvironment are primarily responsible for directing this differentiation.The proportions for each subgroup can fluctuate depending on the manner in which the infection progresses 78,79 .A strong CD4 + response of mixed Th1/Th2 subsets early in the intestinal phase of trichinellosis, is provoked in intestinal mucosa with dominance of Th2 cytokine profile 4 .Increased luminal fluid and mastocytosis with its degranulation, which breaks down epithelial tight junctions, are among the immune-pathological changes that ensue.Lately, TGF-β has recently been discovered to control CD4 + polarization into Th17, boosting the hyper-contractility of intestinal muscles and the propulsive activity that leads to the expulsion of worms 70,78 .
The treatment groups also showed significant increase that represented as significant elevation on GSH level and antioxidant enzymes; GR, GST, CAT and SOD activities especially in mixture group when compared to the infected group.All these antioxidants of pumpkin extract scavenge the free radicals produced after infection to T. spiralis as MDA and protect the gastrointestinal cell membranes from lipid peroxidation so could restore histopathological findings as demonstrated in preceding studies 80,81 .
A crucial enzyme in the process of turning arachidonic acid into prostanoids is cyclooxygenase.Numerous pathological processes, including inflammation, tissue healing, and eventually carcinogenesis, are impacted by isoform cyclo-oxygenase-2 expression 82 .When compared to the positive control group, the immunohistochemistry results of combination therapy showed little immunostaining of COX-2 in both epithelial covering and inflammatory cells in lamina propria.These findings were consistent with those made by Saki et al. 83 who claimed that using E. purpurea can inhibit the expression of the IL-17 gene and the COX -2 enzyme.Additionally, prior research indicated the decrease of COX -2 caused by Echinacea 84,85 .This COX -2 suppression may function as a defense mechanism against T. spiralis infection and may alleviate pathological abnormalities attributed to T. spiralis.This was corroborated by Mulshine et al. 86 who claimed that COX -2 inhibitors are a useful approach for reducing a variety of inflammatory diseases.According to Sadek and El-Aswad 87 and Hamed et al. 88 , COX -2 inhibition plays a helpful part in the management of cryptosporidiosis and Trichinellosis, respectively.
To explore the Sweep stage, we explained the role of TGF-1β and IL-17 in intestinal contractility.Sweep is regulated by the cooperation of a hormone-immune circuit and is correlated with muscular contractions.Mice models have exhibited that expulsion of T. spiralis is related to a vigorous and acute T helper 2 (Th2) CD4 + T-cell response, around one-week p.i. in mice.Steel et al. 70 demonstrated that T. spiralis infected mice display enhanced TGF-1β signaling in intestinal CD + 4 T cells that drives Th17 induction.Th17 response promotes intestinal contractility and "sweep" mechanism of parasites expulsion.A remarked elevation in TGF-1β production in parallel to promoted Th2 responses in T. spiralis mice model around one-week p.i. in mice.Increase TGF-1β is associated with a significant increase in IL-17 secretion in intestinal tissue.Additionally, incubation of gut strips isolated from wild-type mice with IL-17 increased tension, suggesting that IL-17 can promote tension and thus potentially lead to parasite expulsion.Also, IL-17 drive Paneth cells antimicrobial peptide production and IgA secretion in T. spiralis mice model.In addition to that, IL-17 has a direct effect on T. spiralis behavior and epithelial permeability.The same results were recorded in our results, where TGF-1β and IL-17 significantly elevated in pumpkin-treated group and mixture-treated group.
Finally, we could conclude that pumpkin seeds decoction exhibited anthelmintic activity by two mechanisms: inducing weep and sweep processes and stimulating immunity Type 2.
In addition, many active components identified in the extract have anti-parasites effect action.For example, Squalene, the major terpen, occurs in pumpkin decoction.Cárdeno et al. 89 reported that squalene exhibited immunomodulatory effect on lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages and human monocytes and neutrophils.Squalene inhibited the expression of intracellular levels of cytokines (TNF-α, IL-1β, IL-6 and IFN-γ), nitrites, and pro-inflammatory enzymes (iNOS, COX-2, and MPO) and ROS.Also, the two major components of phenols compounds in the pumpkin decoction, gallic acid and chlorogenic acid have properties to help in worm expulsion.Gallic acid normalized the elevated TGF-1β in liver 90 .Also, He et al. 91 reported that gallic acid pretreatment prohibited the elevation of IL-1b and increased IL-10 and TGF-β in LPS-treated animals.Similarly, E. coli 1587 infection strongly promoted the up-regulation of IL-1b, IL-6, IL-10, and TNF-a mRNA transcription levels in mice colons 3 days' post infection.Gallic acid downregulated the upregulations of IL-1b and TNF-α of E. coli 1587 animals, accompanied by elevated TGF-1β mRNA expression level.Exposure to Gallic acid preserved cell integrity and barrier function, and inhibited the transcription levels of IL-1b, with an up-regulation of IL-10 and TGF-b expressions post promotion of ESBL-EAEC-LPS.Chlorogenic acid, an intact intestinal barrier plays an important role in gut-related disease.Chlorogenic acid relieved the intestinal mucosal inflammation via reduction intestinal permeability and elevation intestinal expression of www.nature.com/scientificreports/tight junction proteins on intestinal barrier function in weaned rats challenged with LPS.Chlorogenic acid does its anti-inflammatory activity through suppression the release of some mediators like TNF-α, IL-6 and IL-1β 92 .

Conclusion
The present study demonstrated that pumpkin seeds decoction has anthelmintic effect against Trichinella spiralis in the enteric stage.Pumpkin decoction reduced worm burden in intestine and ameliorated intestine tissue histology.Mixing pumpkin decoction with honey increased its activity to be bitter than albendazole, the reference drug.Pumpkin decoction expelled T. spiralis worms by Weep and Sweep mechanism.Where, pumpkin decoction promoted TGF-1β expression, which controlling mast cell expression of the gut homing integrin alpha E and MMCP-1, essential for the Weep aspects of T. spiralis.Besides, TGF-1β promoted Th17 cells differentiation increasing IL-17 production.IL-17 increased intestine contractility, essential for Sweep aspects of T. spiralis.
The anthelmintic effect of pumpkin decoction may be attributed to its content of terpenes especially squalene, polyphenols, flavonoids, tannins, crude alkaloids, and especially gallic and chlorogenic acid.This study can be generalized to a broader study population and clinical trial as the pumpkin decoction is completely safe during the experimental period.

Figure 1 .
Figure 1.TNF-α of trichinosis mice treated with pumpkin decoction and its mixture with honey.Results are shown as the means ± SE of six duplicates.The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 2 .
Figure 2. TGF-β1 of trichinosis mice treated with pumpkin decoction and its mixture with honey.Results are shown as the means ± SE of six duplicates.The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 3 .
Figure 3. IL-17 of trichinosis mice treated with pumpkin decoction and its mixture with honey.Results are shown as the means ± SE of six duplicates.The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 4 .
Figure 4.Sections of ileum of studied groups; (a,b): uninfected control group showing normal appearance of intestinal layers mucosa comprised in columnar cells (red arrowhead) with goblet cells (wavy arrows), lamina propria (curved arrows); submucosa (green arrow), musculosa (red arrow) and serosa covered with mesothelium (arrowhead).Also, intestinal villi (arrows) with normal architecture and length, shallow intestinal crypts are visible (blue arrow).(c,d): Infected, non-treated group showing malformation of the intestinal villi (arrowhead) and marked inflammation (I) and mature T. spiralis lodged in the intramucosal and in the lumen (arrows).(e,f): Pumpkin decoction treated mice with moderate inflammatory cell infiltrates and slight necrosis could be noticed in villi (curved arrows).(g,h): Honey treated mice showed moderate inflammatory infiltration (I) and necrotic and damaged villi (curved arrows).(i,j): Pumpkin decoction + honey mixture administrated group displayed an improved appearance, with the majority of the villi returning to their typical design with hyperplasia of goblet cells and an apparent decrease in the inflammatory infiltration.(k,l): Albendazole manipulated group exhibited T. spiralis adults in the core of villi (black arrows) with obvious inflammatory infiltration (red arrow), also villi appeared detached and atrophied (arrowheads).Original magnifications were 200 × (Scale-bar: 100 µm), 400× (Scale-bar: 50 µm).Quantitative estimation of mean number of goblet cells from 4 images/sample/group using Image-Pro Plus software is charted down the images.** *p < 0.05 compared to uninfected.** p < 0.05 compared to infected.*p < 0.05 compared to pumpkin.#p < 0.05 compared to honey.$p < 0.05 compared to standard.

Figure 5 .
Figure 5. (a) Ileum tissue of negative control group (GI) showing no immunostaining, (b): infected control group (GII), COX-2 is expressed moderately in the mucosal epithelium and diffusely in inflammatory cells at the center of intestinal villi with moderate expression cells.(c) Subgroup (I) that received pumpkin decoction showing focused expression of COX-2 in inflammatory cells and no expression in epithelial cells.(d): COX-2 expression in the epithelial cells of the subgroup (II) that received honey was moderate, and it was widely expressed in inflammatory cells.(e): COX-2 expression was null in the epithelium and inflammatory cells in subgroup (III) that had combination treatment (f) The subgroup (IV) that only received albendazole revealed that COX-2 was moderately expressed in the cytoplasm of inflammatory cells and the inner core of intestinal villi.Quantitative estimation of immunohistochemical staining based on the determination of the % positive-stained area from 4 images/sample/group using Image-Pro Plus software is charted down the images.***p < 0.05 compared to uninfected.**p < 0.05 compared to infected.*p < 0.05 compared to pumpkin.#p < 0.05 compared to honey.$p < 0.05 compared to standard.Original magnification was 400× (Scale-bar: 50 µm). https://doi.org/10.1038/s41598-024-51616-4 All procedures were conducted in adherence to the Guide for the Care and Use of Laboratory Animals (Eighth edition) of the National Institutes of Health and approved by the Research Ethical Committee for Animal Subject Research at Faculty of Women -Ain Shams University in Egypt under registration No. #ASU/W/Sci-5R/23-2-36 in 2023, and all of them were performed in accordance with ARRIVE guidelines.

Table 3 .
Oxidative stress status of trichinosis mice treated with pumpkin decoction.Results are shown as the means ± SE of six duplicates.The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Table 4 .
Safety profile of trichinosis mice treated with pumpkin decoction and its mixture with honey.Results are shown as the means ± SE of six duplicates.The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Table 5 .
Organoleptic properties and chemical composition of decoction of pumpkin seeds.

Table 6 .
Polyphenolic compounds of the decoction of pumpkin seeds as identified by HPLC.

Table 7 .
Crude lipid composition of pumpkin seeds using GC, fatty acids composition (part A) and unsaponifiable fraction (part B).Significant values are in bold.